Assessment of Fruit Quality and Fruit Morphology in Androgenic Pepper Lines (Capsicum annuum L.)

Abstract

Anther or microspore culture induced haploid and double haploids (DH) are increasingly being utilized by breeders since it can shorten breeding time by achieving complete homozygosity within a single generation. Pepper (Capsicum annuum L.) is one of the most important vegetables, distinguished by its high level of heterozygosity, making the breeding process very laborious and long. Fourteen different DH lines were obtained as a result of anther culture of four parental genotypes. Data from different plant and fruit morphological traits as well as quality traits including vitamin C, dry matter content, total polyphenols and antioxidant activity were collected. A total of 47 different descriptors for fruit morphology and color were characterized using Tomato Analyzer v. 3 software. Findings from this research revealed significant variation of fruit morphology, quality and productivity traits between DH lines and their respective parental genotypes. Among these studied 14 DH lines, 42.9% were superior to the parental genotypes for fruit weight, width, fruit wall thickness, and usable part of the fruits. As compared to parental genotypes, DH lines exhibited higher values for tested fruit quality traits and dry matter content. Multivariate analysis allowed us to identify the trait combination that contributed to the total variation. A total of eight principle components (PCs) explained 95.4% variation with PC1 and PC2 contributing 32.4% and 21.1%, respectively. Pepper androgenesis clearly indicates its usefulness as a well established technique that can allow pepper breeders to save the time and breeding resources by expediting the breeding process. Our research findings prove the advantages of pepper androgenesis to utilize the diversity of pepper genetic resources and development of novel pepper breeding lines to utilize in future breeding.

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Article
Assessment of Fruit Quality and Fruit
Morphology in Androgenic Pepper Lines
(Capsicum annuum L.)
Stanislava Grozeva 1,*, Ivanka Tringovska 1, Amol N. Nankar 2,
Velichka Todorova 1, Dimitrina Kostova 2
1 Maritsa Vegetable Crops Research Institute, Plovdiv 4003, Bulgaria
2 Center of Plant Systems Biology and Biotechnology, Plovdiv 4000, Bulgaria
* Correspondence: Stanislava Grozeva, Email: stanislava_grozeva@abv.bg;
Tel.: +359-032-95-12-96.
ABSTRACT
Anther or microspore culture induced haploid and double haploids (DH)
are increasingly being utilized by breeders since it can shorten breeding
time by achieving complete homozygosity within a single generation.
Pepper (Capsicum annuum L.) is one of the most important vegetables,
distinguished by its high level of heterozygosity, making the breeding
process very laborious and long. Fourteen different DH lines were
obtained as a result of anther culture of four parental genotypes. Data
from different plant and fruit morphological traits as well as quality
traits including vitamin C, dry matter content, total polyphenols and
antioxidant activity were collected. A total of 47 different descriptors for
fruit morphology and color were characterized using Tomato Analyzer v.
3 software. Findings from this research revealed significant variation of
fruit morphology, quality and productivity traits between DH lines and
their respective parental genotypes. Among these studied 14 DH lines,
42.9% were superior to the parental genotypes for fruit weight, width,
fruit wall thickness, and usable part of the fruits. As compared to
parental genotypes, DH lines exhibited higher values for tested fruit
quality traits and dry matter content. Multivariate analysis allowed us to
identify the trait combination that contributed to the total variation. A
total of eight principle components (PCs) explained 95.4% variation with
PC1 and PC2 contributing 32.4% and 21.1%, respectively. Pepper
androgenesis clearly indicates its usefulness as a well established
technique that can allow pepper breeders to save the time and breeding
resources by expediting the breeding process. Our research findings
prove the advantages of pepper androgenesis to utilize the diversity of
pepper genetic resources and development of novel pepper breeding
lines to utilize in future breeding.
KEYWORDS: pepper; anther culture; double haploids; total polyphenols;
Tomato Analyzer
Open Access
Received: 06 December
2019
Accepted: 22 January
2020
Published:
23 January 2020
Copyright © 2020
by the
author(s). Licensee Hapres,
London, United Kingdom. This is
an open access article distributed
under the terms and conditions
of Creative Commons Attribution
4.0 International License
.
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Crop Breeding, Genetics and Genomics 2 of 13
INTRODUCTION
Pepper is an economically important crop from the Solanaceae family
characterized by wide variation for fruit color, shape and size. In recent
years, studies have focused on to fruit quality traits with enhanced
antioxidant concentrations and health-promoting properties [1].
Biodiversity using advanced cytological and tissue culture methods has
been proven effective to improve pepper germplasm with novel value
added traits. Production of haploids and double haploids (DHs) is an
important plant-breeding tool that allows rapid recovery of unique
homozygous genetic recombinants, and quick detection of recessive
mutations. Double haploid lines are immensely valuable to speed up the
breeding process. They have been identified as essential breeding
material for crop improvement due to its proven practical applications
for the development of hybrids that display maximum heterosis and/or
improved traits [2,3].
Anther culture is the most applied method used in pepper to obtain
haploids and DH plants. Success of this technique is determined by
numerous factors such as genotype, physiological status of donor plant,
pollen development stage, culture media composition, anther
pretreatments and other unknown factors [4–6]. Spontaneous or induced
genome doubling of haploid allows development of fully homozygous
plants with unique genetic recombinants [7].
The morphological evaluation of DH lines allows the confirmation of
genetic homogeneity of a single line; it also makes it possible to present
the diversity across different lines obtained in anther culture [8,9]. This
variation between DH lines reflects the genetic diversity of microspores,
which results from random gene segregation in meiosis, and is one of the
most important conditions of in vitro androgenesis practical applicability
to plant breeding [10]. On the other hand, studies associated with quality
traits including sugar, ascorbic acid or vitamin C content, total
polyphenols as well as antioxidant properties of DH lines could also be
helpful for selecting the regenerants [11]. Inclusion of this genetic
diversity in pepper breeding programs leads to selection of individuals
with valuable combination of agronomic and fruit quality traits [12,13].
Hence this study was designed to evaluate fruit quality characters and
fruit morphology of 14 diverse androgenic pepper lines.
MATERIALS AND METHODS
The experimental work was carried out during 2018–2019 at Maritsa
Vegetable Crops Research Institute, Plovdiv, Bulgaria. Fourteen DH lines
obtained as a result of self-pollination of anther-derived regenerants and
four parental genotypes—one hybrid “202” and three varieties—“Slonovo
uvo”, “Stryama” and “Zlaten medal 7”. Three of the evaluated lines
originated from hybrid “202”, two lines from variety “Slonovo uvo”, five
lines from variety “Stryama” and four lines from variety “Zlaten medal
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Crop Breeding, Genetics and Genomics 3 of 13
7”. The plants were grown under field conditions with furrow surface
irrigation in a 70/15 cm scheme. The DH lines and parental genotypes
were evaluated during two consecutive years in two replications. Each
replication consisted of 10 plants. The fruits were harvested at maturity.
Plant productivity (g), average fruit weight (g), fruit length (cm), fruit
width (cm), usable part of the fruit (g) and fruit wall thickness (mm) were
determined. These traits were evaluated on five plants per replication
and three fruits per plants.
Eight fruits per genotype were prepared for high-throughput
phenotyping using Tomato Analyzer (TA) software. Fruits were cleaned,
cut into longitudinal and latitudinal sections, scanned at 300 dpi and
analyzed using TA for a total of 47 different fruit morphometric [14] and
colorimetric traits [15].
A sample of ten randomly selected fruits from each genotype were
used for analysis of the following fruit quality traits: dry matter (DM),
ascorbic acid or vitamin C (Vit C) content, Total polyphenols (TP) and
ferric-reducing antioxidant power (FRAP). Fruits were rinsed three times
with distilled water and were wiped. Half of the pericarp was freshly
homogenized to juice and used for analysis of dry matter (by oven drying
at 105 °C to a constant weight) and vitamin C content (by Tillman’s
reaction [16]). Half of the pericarp was lyophilized, powdered and used
for analyzes of total polyphenols and antioxidant activity. Total
polyphenols and ferric-reducing antioxidant power (FRAP) extraction
procedures were performed according to the optimized method
described by Atanasova et al. [17]. Total polyphenols were quantified
according to the Singleton and Rossi [18] method. The FRAP antioxidant
activity was measured following the procedure originally described by
Benzie and Strain [19].
Statistical Analysis
The fruit quality data were analyzed using analysis of variance
(ANOVA) and post-hoc Duncan test to identify between accession
differences. Fruit image data were transformed by log transformation
and were statistically analyzed using ggplot2 package of R program.
Correlation of fruit morphometric and colorimetric traits was
determined by multivariate technique of principal component analysis
(PCA). The correlation and PCA were performed based on the basis of the
average values of the investigated parameters. The PCA was used to
determine between accession variation and various PCA parameters
were estimated using ggplot2, missMDA, FactoMineR, and Factoextra R
packages.
RESULTS
The results from manual measurements of fruit morphology and plant
productivity showed significant phenotypic homogeneity within
androgenic lines and considerable variation for main fruit characters
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Crop Breeding, Genetics and Genomics 4 of 13
and productivity among different androgenic lines of same background
(Table 1). The lines originating from F1 “202” were distinguished with
higher values of the measured fruit morphological traits in comparison
to the parental genotype, while the highest productivity, Vit C, dry
matter, and antioxidant activity was observed in line “211”. The highest
value of TP was measured in line “212” (Table 2). The statistical analysis
of the lines deriving from variety “Slonovo uvo” showed that line “214”
exceeded the parental genotype for studied fruit morphology traits
(Figure 1A,B). In terms of Vit C content, TP, and antioxidant activity, the
higher value was measured in both androgenic lines from variety
“Slonovo uvo”.
Table 1. Morphological evaluation of fruit and productivity in androgenic lines and four parental
genotypes.
Line
Fruits Characters
Productivity/
P
lant (g)
Fruit
/
P
lant No.
W
eight
(g)
L
ength
(cm)
W
idth
(cm)
W
all Thickness
(mm)
U
sable Part
(g)
2
11
141.3
a
14.0
ns
6.2
ab
6.5
a
124.5
a
2047
a
15
ns
212
127.0
a
14.4
ns
6.4
a
6.
5 a
108.3
a
1398
b
12
ns
213
125.8
a
1
5.1 ns
6.6
a
6.
5 a
110.1
a
1449
b
12
ns
F1
202
90.8
b
13.8
ns
5.0
b
4.0
b
83
.3 b
1150
b
12
ns
214
201.5
a
15.0
a
7.5
a
9.1
a
181.5
a
2043
a
11
ns
215
146.0
b
13.0
b
6.5
b
5.1
b
130.5
b
1414
b
11
ns
S. uvo
105.6
b
13.6
ab
6.4
b
5.5
b
92.0
b
1193
b
11
ns
216
100.1
ab
11.9
ab
5.1
a
5.5
ns
85.2
a
1166
ns
12
ab
217
86.3
bc
10.4
b
5.0
a
4.9
ns
74.6
ab
1141
ns
13
ab
218
77.7
c
11.0
b
4.9
a
4.9
ns
64.2
b
1415
ns
18
a
219
86.1
bc
10.8
b
5.1
a
4.9
ns
71.3
ab
1250
ns
15
ab
220
106.0
a
13.0
a
5.3
a
5.1
ns
89.0
a
1190
ns
12
b
Stryama
76.4
c
11.5
ab
4.5
b
4.0
ns
65.7
b
1193
ns
17
a
221
70.7
ns
12.1
ns
4.4
ns
4.7
a
59.4
ns
1237
ab
17
ab
222
85.7
ns
13.7
ns
4.8
ns
4.7
a
72.4
ns
1105
b
14
b
223
80.4
ns
13.4
ns
4.8
ns
4.0
ab
68.4
ns
1296
ab
17
ab
224
80.3
ns
12.1
ns
4.9
ns
4.0
ab
70.2
ns
1418
a
18
a
Z. medal
77.8
ns
14.3
ns
4.7
ns
3.0
b
67.1
ns
1303
ab
17
ab
a–c: p ≤ 0.05, Duncan’s Multiple Range Test; ns: not significant.
Among androgenic lines originated from variety “Stryama”, the
highest fruit weight, length and width were recorded in line “220”
followed by line “216”. The Vit C was found highly variable among
androgenic lines and donor genotype “Stryama”. The highest Vit C
content was recorded in line “218” (70.2 ng/100 g fresh weight (FW)),
while the least in line “220” (39.2 ng/100 g FW). Among the androgenic
lines, no significant differences in DM and TP content were seen, but the
measured values were higher than those in the control. The FRAP
antioxidant activity varied from 6.0 µmol Fe2+/g FW (line “220”) to 8.7
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Crop Breeding, Genetics and Genomics 5 of 13
µmol Fe2+/g FW (line “216”) and 4.5, µmol Fe2+/g FW for variety
“Stryama”.
Table 2. Evaluation of same fruit quality traits and dry matter content in androgenic pepper lines and
parental genotypes.
Line
Vit
C,
ng/100 g FW
Dry matter
,
%
Total
Polyphenols,
mg GAE/100 g FW
FRAP,
µmol Fe
2+
/g FW
211
190.1
10.83
143.8
13.5
212
167.8
10.32
154.5
11.9
213
136.1
10.59
132.8
8.3
F1 202
-
-
-
-
214
192.
0
11
.73
193.
0
12
.1
215
237.
7
12
.17
178.
6
15.
9
S. uvo
151.9
11.28
153.6
8.0
216
40.8
7.38
150.1
8.7
217
55.5
7.36
148.5
7.8
218
70.2
7.90
151.6
6.9
219
35.9
7.50
120.4
6.6
220
21.2
7.06
131.3
6.0
Stryama
39.2
6.05
105.2
4.5
221
106.1
7.59
77
.6
4.
6
222
52.2
7.41
62.
8
3.
7
223
84.9
7.25
73.
4
4.
3
224
106.1
8.03
85.
4
5.
5
Z. medal
24.2
7.23
86.4
3.2
The lines originated from variety “Zlaten medal 7” did not show
higher values of the fruit morphology traits, but lines “221” and “224”
showed 2.5-fold increase in Vit C content (Figure 1C,D). Only line “224”
distinguished by the DM with the value over 8.0%. Data showed that the
TP content was higher in the donor genotype “Zlaten medal 7”. Highest
FRAP antioxidant activity of 5.5 µmol Fe2+/g FW was recorded in line
“224”, followed by line “221” 4.6 µmol Fe2+/g FW least was reported in
donor genotype “Zlaten medal 7”.
Multivariate Principal component analysis (PCA) was employed to
detect and identify the trait combination that most contributed to the
total cumulative variation. A total of 17 principal components were
identified during PCA (Figure 2); however, we utilized factor analysis to
identify those principal components that had an eigan value of >1 (Table
3) and eight major PCs explained around 95.4% variation to the total
variation. The TA descriptors individual PC1 to PC8 contributed 32.4%,
21.1%, 14.1%, 8.5%, 7.8%, 4.6%, 4.4%, and 2.5% variation, respectively
(Figure 2 and Table 3).
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Crop Breeding, Genetics and Genomics 6 of 13
(A)
(B)
(C)
(D)
Figure 1. Androgenic pepper plants (A) Pepper plant from line “214” (B) Scanned fruits from line “214” (C)
Pepper plant from line “221” (D) Scanned fruits from line “221”.
Table 3. Eigenvalues, % variance, and % cumulative variance as explained by extracted factors in factor
analysis.
Component Eigan Values % Variance % Cumulative Variance
1 15.2 32.4 32.4
2 9.9 21.1 53.5
3 6.6 14.1 67.6
4 4.0 8.5 76.1
5 3.7 7.8 83.9
6 2.2 4.6 88.5
7 2.0 4.4 92.9
8 1.2 2.5 95.4
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Crop Breeding, Genetics and Genomics 7 of 13
Figure 2. PCA variance plot displaying percent variation contributed by each principle component. The
line with circle indicates the cumulative variation of 1–17 components, and the line with square indicates
variation explained by an individual principle component.
The PCA accession and feature biplot displayed that all DH lines were
spread across all four quadrants (Figure 3); however, DH lines derived
from a specific variety populated in specific quadrants. Double haploid
lines “221”, “222” derived from “Zlaten medal 7” as well as parental
genotypes of 20 (“Zlaten medal 7”) were limited to quadrant 2 (negative
quadrant of PC1 and positive quadrant of PC2), while DH lines “223” and
“224” were scattered in quadrant 1 (positive quadrant of PC1 and PC2),
and quadrant 3 (negative quadrant of PC1 and PC2), respectively. Double
haploid lines “211”, “212”, “213” derived from the F1 hybrid “202” were
distinctly separated in quadrant 1 (Figure 3). Double haploid lines “214”
and “215” derived from “Slonovo uvo” populated in the positive quadrant
of PC1 and negative quadrant of PC2 (quadrant 4), whereas parental
genotype of 47 (“Slonovo uvo”) was populated in quadrant 3. Double
haploid lines “216”, “217”, “218”, and “219” derived from “Stryama” were
populated in quadrant 3 while parental genotype 23 (“Stryama”) was
located in quadrant 2. PC1 exhibited contribution by color, proximal fruit
end shape, internal eccentricity whereas fruit size, fruit blockiness, and
distal fruit end shape descriptors contributed to PC2 (Figure 3).
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Crop Breeding, Genetics and Genomics 8 of 13
Figure 3. The relationship of the first (PC1) and second (PC2) components in the analysis of 14 pepper DH
lines and three parental genotypes.
DISCUSSION
In recent years, the application of anther culture as a breeding
method has significantly increased due to its advantage in speeding up
the breeding process. By anther and pollen culture, DH plants can be
produced within a year in comparison to prolong inbreeding, which
usually takes more than six years.
Pepper belongs to a recalcitrant species characterized by low
frequency of embryo induction from anthers and low rate of subsequent
conversion to normal plant-regenerants [2,20]. Despite this difficulty, the
number of studies concerning the practical application in different
Capsicum species has steadily increased in recent years [21–25]. Different
studies indicate that positive changes in plant productivity, fruit
morphology, resistance to viruses and pests can be induced with anther
culture [26–28]. The findings from the present study showed significant
variation for main fruit characters and productivity among androgenic
lines of same origin. These differences may be due to the somatic changes
that occur in the pollen grain DNA, which likely results into new gene
combinations obtained during meiosis [10,29]. The phenotypic diversity
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Crop Breeding, Genetics and Genomics 9 of 13
reflects the genetic variability of microspores they originated from and
essential characteristics of androgenic populations, which greatly
facilitates the selection of plants valuable for breeders [8,29]. Nowaczyk
et al. [30] indicated that 74% of studied androgenic plants were different
compared to the donor genotype for ripe fruit color, pericarp thickness,
and fruit taste. Other nine diploid androgenic plants showed differences
from the mother genotypes for fruit weight, fruit wall thickness, and dry
matter content. Other investigations also reported variation in plant and
fruits characters between androgenic lines of the same origin [8,29].
Shrestha et al. [29] suggested that the differences between DH lines may
be due to the results of naturally produced variation, mutation during
anther culture, but also has been attributed to residual heterozygosity in
a donor plants for anther culture.
In the current study, androgenic lines derived from hybrid “202” and
“Slonovo uvo” were distinguished with the heaviest fruits compared to
the parental genotypes. Average fruit weight was also higher in the DH
lines originated from varieties “Stryama” and “Zlaten medal 7”, but the
differences were not substantial. Fruit wall thickness was the trait by
which all DH lines were superior to the parental genotypes. Moreover,
our data showed higher productivity per plant in six DH lines compared
to the donor genotypes. Similar results with improved fruit morphology
characteristics were obtained in other studies [8,12,13]. On the other
hand, Luitel et al. [31] reported that plant and fruit characters in DH
plants were lower than the standard varieties but should not be omitted
in the process of DHs evaluation. After detailed study of
agromorphological and molecular traits in eight double haploid pepper
lines, it was established that two of them had higher yield [32]. As
pointed out in different investigations, DH lines can be used as a tool for
enrichment of biodiversity and fast development of valuable pepper
genotypes for future breeding [11,13].
Pepper fruits are rich in biologically active substances such as
carotenoids, vitamins, flavonoids [33]. However, the variation of the fruit
biochemical composition is mainly affected by the genotype [34]. The
results of the current study showed a wide variation in Vit C, dry matter
content, TA, and FRAP among studied DH lines. Generally, the highest
value of Vit C content was obtained in red colored fruits. Nevertheless, in
lines “221” and “224” the Vit C content was 2.5 fold higher than the
control “Zlaten medal 7”. Luitel and Kang [11] observed the higher value
of Vit C in yellow and orange colored fruits compared to red genotypes.
The authors suggested that the variation could be due to the genotype,
maturity stage, and environmental conditions. The variation in dry
matter content among the studied DH lines and initial genotypes was
variable from 6.05% to 12.17%. The same results with variation in dry
matter content in different lines with androgenic origin between 7.85%
and 13.0% was also reported [8]. The highest value of total polyphenols
was measured in line “214” (193.0 mg GAE/100 g FW) and the least in line
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Crop Breeding, Genetics and Genomics 10 of 13
“222” (62.8 mg GAE/100 g FW). In contrast, Luitel and Kang [11] reported
the highest content of gallic acid of 18.8 mg g−1 of FW in androgenic line
MY-3. In all studied DH lines, antioxidant activity was higher than the
initial genotypes and varied widely. The results of the present study are
in accordance with previous research, which indicated that antioxidant
activity varied among pepper varieties and DH lines [11,35].
Furthermore, higher polyphenol content positively correlated with
antioxidant activity [35].
CONCLUSIONS
The results of the present study showed that androgenic pepper lines
were different from the parental genotype. Variation among androgenic
lines of same origin also was observed. On the basis of morphological
and fruit quality evaluation, pepper lines “211”, “214”, “215” and “224”,
superior to the parental genotypes, were selected and will be further
utilized in future breeding programs. Findings of this study demonstrate
that the application of anther culture can assist in obtaining genetic
variation and transgressive traits. The accessible genetic diversity of
pepper can be resourceful in development of novel breeding lines with
improved quantitative and qualitative fruit quality traits.
AUTHOR CONTRIBUTIONS
SG designed the manuscript. SG, IT, AN and DK carried out the
experimental work. SG and IT analyzed the results. AN designed the data
of TA. VT provided the seeds from parental genotypes. DK supervised the
process. All authors read and corrected the manuscript.
CONFLICTS OF INTEREST
The authors declare that there is no conflict of interest.
FUNDING
This study was funded by the National Science Fund of Bulgaria
[Grant DN06/4] and the financial support by Horizon 2020 PlantaSYST
project under Grant Agreement No 739582.
ACKNOWLEDGMENTS
We would like to acknowledge the efforts of technical staff of Plant
Tissue Culture Laboratory of Maritsa Vegetables Crops Research Institute
(MVCRI) in conducting the experiemnts to generate the DH lines.
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How to cite this article:
Grozeva S, Tringovska I, Nankar AN, Todorova V, Kostova D. Assessment of Fruit Quality and Fruit Morphology
in Androgenic Pepper Lines (Capsicum annuum L.). Crop Breed Genet Genom. 2020;2(1):e200005.
https://doi.org/10.20900/cbgg20200005
Crop Breed Genet Genom. 2020;2(1):e200005. https://doi.org/10.20900/cbgg20200005